Increased Src kinase level results in increased protein tyrosine phosphorylation in scrapie-infected neuronal cell lines

We have studied how prion infection may affect the Src kinase activity in three different neuronal cell lines, ScGT1 and ScN2a, where ScGT1 were generated in our laboratory. By immunoblotting, using clone 28 - a monoclonal antibody recognizing active Src, we have found a 32+/-6.3% and 75+/-7.7% elevation in Src activity in ScGT1 and ScN2a cells, respectively, compared to uninfected cells. Immunocomplex in vitro kinase assay confirmed the increased Src activity. The increased Src kinase activity in scrapie-infected cells was further shown to correlate to an increased level of Src protein. In addition, an important increase in the protein tyrosine phosphorylation signal was observed in ScGT1 and ScN2a cells, which was further shown to be Src-dependent, as treatment with PP2 - a Src family kinase specific inhibitor, reversed the protein tyrosine phosphorylation profile. Abnormal Src-kinase activation and subsequent protein tyrosine phosphorylation may be key elements in the neuropathology of the prion diseases.     

Src and Pyk2 mediate G-protein-coupled receptor activation of epidermal growth factor receptor (EGFR) but are not required for coupling to the mitogen-activated protein (MAP) kinase signaling cascade

The epidermal growth factor receptor (EGFR) and the non-receptor protein tyrosine kinases Src and Pyk2 have been implicated in linking a variety of G-protein-coupled receptors (GPCR) to the mitogen-activated protein (MAP) kinase signaling cascade. In this report we apply a genetic strategy using cells isolated from Src-, Pyk2-, or EGFR-deficient mice to explore the roles played by these protein tyrosine kinases in GPCR-induced activation of EGFR, Pyk2, and MAP kinase. We show that Src kinases are critical for activation of Pyk2 in response to GPCR-stimulation and that Pyk2 and Src are essential for GPCR-induced tyrosine phosphorylation of EGFR. By contrast, Pyk2, Src, and EGFR are dispensable for GPCR-induced activation of MAP kinase. Moreover, GPCR-induced MAP kinase activation is normal in fibroblasts deficient in both Src and Pyk2 (Src-/-Pyk2-/- cells) as well as in fibroblasts deficient in all three Src kinases expressed in these cells (Src-/-Yes-/-Fyn-/- cells). Finally, experiments are presented demonstrating that, upon stimulation of GPCR, activated Pyk2 forms a complex with Src, which in turn phosphorylates EGFR directly. These experiments reveal a role for Src kinases in Pyk2 activation and a role for Pyk2 and Src in tyrosine phosphorylation of EGFR following GPCR stimulation. In addition, EGFR, Src family kinases, and Pyk2 are not required for linking GPCRs with the MAP kinase signaling cascade.     

Src kinase mediates the regulation of phospholipase C-gamma activity by glycosphingolipids

Glucosylceramide-based glycosphingolipids have been previously demonstrated to regulate negatively the formation of inositol 1,4,5-trisphosphate by phospholipase C-gamma1. In the present study, the depletion of endogenous glucosylceramide by D-t-EtDO-P4 in cultured ECV304 cells induced autophosphorylation of Src kinase at tyrosine residue 418 within the catalytic loop and dephosphorylation of Src kinase at tyrosine residues 529 within the carboxyl-terminal regulatory region. Phosphotransferase activities of Src kinase were also induced in the glucosylceramide-depleted cells. c-Src kinase activity and phosphorylations at Src Tyr-418 and epidermal growth factor (EGF) receptor Tyr-1068 were significantly enhanced by bradykinin in response to 100 nm D-t-EtDO-P4 compared with control cells. The phosphorylation and dephosphorylation on Tyr-418 and Tyr-529 residues of c-Src were reversed by treatment of 4-amino-5-(4-chlorophenyl)-7-t-butyl(pyrazolo)[3,4-d]pyrimidine (PP2), an inhibitor of Src kinase, in control cells. Glucosylceramide-depleted cells resisted treatment with PP2, and both phosphorylation of Tyr-418 and dephosphorylation of Tyr-529 induced by depletion of glucosylceramide were maintained. Compared with untreated cells, tyrosine phosphorylation of phospholipase C-gamma1 was enhanced by EGF stimulation in glucosylceramide-depleted cells, associated with enhanced tyrosine phosphorylation of the EGF receptor at Tyr-1068 and Tyr-1086 stimulated by EGF. The Src inhibitor, PP2, significantly blocked EGF-induced tyrosine phosphorylation of phospholipase C-gamma1 in control cells, whereas in glucosylceramide-depleted cells, suppression of Src kinase activity by PP2 toward EGF-induced tyrosine phosphorylation of phospholipase C-gamma1 was less significant. Thus the activation of Src kinase by depletion of glucosylceramide-based glycosphingolipids in cultured ECV304 cells is a critical up-stream event in the activation of phospholipase C-gamma1.     

The inhibition of tube formation in a collagen-fibrinogen, three-dimensional gel by cleaved kininogen (HKa) and HK domain 5 (D5) is dependent on Src family kinases

Cleaved high molecular weight kininogen (HKa), as well as its domain 5 (D5), inhibits migration and proliferation induced by angiogenic factors and induces apoptosis in vitro. To study its effect on tube formation we utilized a collagen-fibrinogen, three-dimensional gel, an in vitro model of angiogenesis. HKa, GST-D5 and D5 had a similar inhibitory effect of tube length by 90+/-4.5%, 86+/-5.5% and 77+/-12.9%, respectively. D5-derived synthetic peptides: G440-H455 H475-H485 and G486-K502 inhibited tube length by 51+/-3.7%, 54+/-3.8% and 77+/-1.7%, respectively. By a comparison of its inhibitory potency and its sequences, a functional sequence of HKa was defined to G486-G496. PP2, a Src family kinase inhibitor, prevented tube formation in a dose-dependent manner (100-400 nM), but PP3 at 5 microM, an inactive analogue of PP2, did not. HKa and D5 inhibited Src 416 phosphorylation by 62+/-12.3% and 83+/-6.1%, respectively. The C-terminal Src kinase (Csk) inhibits Src kinase activity. Using a siRNA to Csk, expression of Csk was down-regulated by 86+/-7.0%, which significantly increased tube length by 27+/-5.8%. The addition of HKa and D5 completely blocked this effect. We further showed that HKa inhibited Src family kinase activity by disrupting the complex of uPAR, alphavbeta3 integrin and Src. Our results indicate that the anti-angiogenic effect of HKa and D5 is mediated at least in part through Src family kinases and identify a potential novel target for therapeutic inhibition of neovascularization in cancer and inflammatory arthritis.     

Discovery and preliminary structure-activity relationship studies of novel benzotriazine based compounds as Src inhibitors

We report the discovery and preliminary SAR studies of a series of structurally novel benzotriazine core based small molecules as inhibitors of Src kinase. To the best of our knowledge, benzotriazine template based compounds have not been reported as kinase inhibitors. The 3-(2-(1-pyrrolidinyl)ethoxy)phenyl analogue (43) was identified as one of the most potent inhibitors of Src kinase.     

c-Src tyrosine kinase binds the beta 2-adrenergic receptor via phospho-Tyr-350, phosphorylates G-protein-linked receptor kinase 2, and mediates agonist-induced receptor desensitization

The nonreceptor tyrosine kinase Src has been implicated in the switching of signaling of beta2-adrenergic receptors from adenylylcyclase coupling to the mitogen-activated protein kinase pathway. In the current work, we demonstrate that Src plays an active role in the agonist-induced desensitization of beta2-adrenergic receptors. Both the expression of dominant-negative Src and treatment with the 4-amine-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) inhibitor of Src kinase activity blocks agonist-induced desensitization. Agonist triggers tyrosine phosphorylation of the beta2-adrenergic receptor and recruitment and activation of Src. Because phosphorylation of the Tyr-350 residue of the beta2-adrenergic receptor creates a conditional, canonical SH2-binding site on the receptor, we examined the effect of the Y350F mutation on Src phosphorylation, Src recruitment, and desensitization. Mutant beta2-adrenergic receptors with a Tyr-to-Phe substitution at Tyr-350 do not display agonist-induced desensitization, Src recruitment, or Src activation. Downstream of binding to the receptor, Src phosphorylates and activates G-protein-linked receptor kinase 2 (GRK2), a response obligate for agonist-induced desensitization. Constitutively active Src increases GRK phosphorylation, whereas either expression of dominant-negative Src or treatment with the PP2 inhibitor abolishes tyrosine phosphorylation of GRK and desensitization. Thus, in addition to its role in signal switching to the mitogen-activated protein kinase pathway, Src recruitment to the beta2-adrenergic receptor and activation are obligate for normal agonist-induced desensitization.     

Protein kinase C-Fyn kinase cascade mediates the oleic acid-induced disassembly of neonatal rat cardiomyocyte adherens junctions

Oleic acid (OA) affects assembly of gap junctions in neonatal cardiomyocytes. Adherens junction (AJ) regulates the stability of gap junction integrity; however, the effect of OA on AJ remains largely unexplored. The distribution of N-cadherin and catenins at cell–cell junction was decreased by OA. OA induced activation of protein kinase C(PKC)-α and -? and Src family kinase, and all three kinases were involved in the oleic acid-induced disassembly of the adherens junction, since it was blocked by pretreatment with Gö6976 (a PKCα inhibitor), ?V1–2 (a PKC? inhibitor), or PP2 (a Src family kinase inhibitor). Src family kinase appeared to be the downstream of PKC-α and -?, as blockade of either PKC-α or -? activity prevented the OA-induced activation of Src family kinase. Immunoprecipitation analyses showed that OA activated Fyn and Fer. OA promoted the association of p120 catenin/β-catenin with Fyn and Fer and caused increased tyrosine phosphorylation of p120 catenin and β-catenin, resulting in decreased binding of the former to N-cadherin and of the latter to α-catenin. Pretreatment with PP2 abrogated this OA-induced tyrosine phosphorylation of p120 catenin and β-catenin and restored the association of N-cadherin with p120 catenin and that of β-catenin with α-catenin. In conclusion, these results show that OA activates the PKC-Fyn signaling pathway, leading to the disassembly of the AJ. Therefore, inhibitors of PKC-α/-? and Src family kinase are potential candidates as cardioprotection agents against OA-induced heart injury during ischemia-reperfusion.     

Oxidative refolding of lysozyme in trifluoroethanol (TFE) and ethylene glycol: interfering role of preexisting alpha-helical structure and intermolecular hydrophobic interactions

We investigated the effect of aldosterone on Src kinase. In the kidney cell line, M-1 aldosterone leads to a >2-fold transient activation of Src kinase seen as early as 2 min after aldosterone administration. Maximal Src kinase activation was measured at an aldosterone concentration of 1 nM. In parallel to activation, autophosphorylation at Tyr-416 of Src kinase increased. Src kinase activation was blocked by spironolactone. Aldosterone led to increased association of Src with HSP84. Furthermore, rapamycin blocked aldosterone-induced Src activation. We conclude that Src activation by aldosterone is mediated through the mineralocorticoid receptor and HSP84.     

A FAK/Src chimera with gain-of-function properties promotes formation of large peripheral adhesions associated with dynamic actin assembly

Formation of a complex between the tyrosine kinases FAK and Src is a key integrin-mediated signaling event implicated in cell motility, survival, and proliferation. Past studies indicate that FAK functions in the complex primarily as a "scaffold," acting to recruit and activate Src within cell/matrix adhesions. To study the cellular impact of FAK-associated Src signaling we developed a novel gain-of-function approach that involves expressing a chimeric protein with the FAK kinase domain replaced by the Src kinase domain. This FAK/Src chimera is subject to adhesion-dependent activation and promotes tyrosine phosphorylation of p130Cas and paxillin to higher steady-state levels than is achieved by wild-type FAK. When expressed in FAK -/- mouse embryo fibroblasts, the FAK/Src chimera resulted in a striking cellular phenotype characterized by unusual large peripheral adhesions, enhanced adhesive strength, and greatly reduced motility. Live cell imaging of the chimera-expressing FAK -/- cells provided evidence that the large peripheral adhesions are associated with a dynamic actin assembly process that is sensitive to a Src-selective inhibitor. These findings suggest that FAK-associated Src kinase activity has the capacity to promote adhesion integrity and actin assembly.     

Identification and functional characterization of an Src homology domain 3 domain-binding site on Cbl

Cbl is an adaptor protein and ubiquitin ligase that binds and is phosphorylated by the nonreceptor tyrosine kinase Src. We previously showed that the primary interaction between Src and Cbl is mediated by the Src homology domain 3 (SH3) of Src binding to proline-rich sequences of Cbl. The peptide Cbl RDLPPPPPPDRP(540-551), which corresponds to residues 540-551 of Cbl, inhibited the binding of a GST-Src SH3 fusion protein to Cbl, whereas RDLAPPAPPPDR(540-551) did not, suggesting that Src binds to this site on Cbl in a class I orientation. Mutating prolines 543-548 reduced Src binding to the Cbl 479-636 fragment significantly more than mutating the prolines in the PPVPPR(494-499) motif, which was previously reported to bind Src SH3. Mutating Cbl prolines 543-548 to alanines substantially reduced Src binding to Cbl, Src-induced phosphorylation of Cbl, and the inhibition of Src kinase activity by Cbl. Expressing the mutated Cbl in osteoclasts induced a moderate reduction in bone-resorbing activity and increased amounts of Src protein. In contrast, disabling the tyrosine kinase-binding domain of full-length Cbl by mutating glycine 306 to glutamic acid, and thereby preventing the previously described binding of the tyrosine kinase-binding domain to the Src phosphotyrosine 416, had no effect on Cbl phosphorylation, the inhibition of Src activity by full-length Cbl, or bone resorption. These data indicate that the Cbl RDLPPPP(540-546) sequence is a functionally important binding site for Src.     

Drosophila C-terminal Src kinase negatively regulates organ growth and cell proliferation through inhibition of the Src, Jun N-terminal kinase, and STAT pathways

Src family kinases regulate multiple cellular processes including proliferation and oncogenesis. C-terminal Src kinase (Csk) encodes a critical negative regulator of Src family kinases. We demonstrate that the Drosophila melanogaster Csk ortholog, dCsk, functions as a tumor suppressor: dCsk mutants display organ overgrowth and excess cellular proliferation. Genetic analysis indicates that the dCsk(-/-) overgrowth phenotype results from activation of Src, Jun kinase, and STAT signal transduction pathways. In particular, blockade of STAT function in dCsk mutants severely reduced Src-dependent overgrowth and activated apoptosis of mutant tissue. Our data provide in vivo evidence that Src activity requires JNK and STAT function.     

beta -Arrestin-mediated recruitment of the Src family kinase Yes mediates endothelin-1-stimulated glucose transport

The insulin and the endothelin type A (ETA) receptor both can couple into the heterotrimeric G protein alpha(q/11) (Galpha(q/11)), leading to Galpha(q/11) tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and subsequent stimulation of glucose transport. In this study, we assessed the potential role of Src kinase in ET-1 signaling to glucose transport in 3T3-L1 adipocytes. Src kinase inhibitor PP2 blocked ET-1-induced Src kinase activity, Galpha(q/11) tyrosine phosphorylation, and glucose transport stimulation. To determine which Src family kinase member was involved, we microinjected anti-c-Src, -c-Fyn, or -c-Yes antibody into these cells and found that only anti-c-Yes antibody blocked GLUT4 translocation (70% decreased). Overexpression or microinjection of a dominant negative mutant (K298M) of Src kinase also inhibited ET-1-induced Galpha(q/11) tyrosine phosphorylation and GLUT4 translocation. In co-immunoprecipitation experiments, we found that beta-arrestin 1 associated with the ETA receptor in an agonist-dependent manner and that beta-arrestin 1 recruited Src kinase to a molecular complex that included the ETA receptor. Microinjection of beta-arrestin 1 antibody inhibited ET-1- but not insulin-stimulated GLUT4 translocation. In conclusion, 1) the Src kinase Yes can induce tyrosine phosphorylation of Galpha(q/11) in response to ET-1 stimulation, and 2) beta-arrestin 1 and Src kinase form a molecular complex with the ETA receptor to mediate ET-1 signaling to Galpha(q/11) with subsequent glucose transport stimulation.     

Bone-targeted Src kinase inhibitors: novel pyrrolo- and pyrazolopyrimidine analogues

Src tyrosine kinase is a therapeutic target for bone diseases that has been validated by gene knockout studies. Furthermore, in vitro cellular studies implicate that Src has a positive regulatory role in osteoclasts and a negative regulatory role in osteoblasts. The potential use of Src inhibitors for osteoporosis therapy has been previously shown by novel bone-targeted ligands of the Src SH2 (e.g., AP22408) and non-bone-targeted, ATP-based inhibitors of Src kinase. Significant to this study, compounds 2-12 exemplify novel analogues of known pyrrolopyrimidine and pyrazolopyrimidine template-based Src kinase inhibitors that incorporate bone-targeting group modifications designed to provide tissue (bone) selectivity and diminished side effects. Accordingly, we report here the structure-based design, synthetic chemistry and biological testing of these compounds and proof-of-concept studies thereof.     

Src kinase activity is essential for osteoclast function

Deletion of the c-src gene impairs osteoclast bone resorbing activity, causing osteopetrosis. Although it has been concluded that restoring only the Src adaptor function at least partly rescues the cell attachment and skeletal phenotypes, the contribution of Src kinase activity remains controversial. Src forms a complex with Pyk2 and Cbl after adhesion-induced stimulation of alpha(V)beta(3) integrin. To demonstrate the importance of the Pyk2-Src association in osteoclasts and to distinguish the contributions of the Src adaptor and kinase activities in cytoskeletal organization and osteoclast function, we expressed mutants of Src and Pyk2 in osteoclasts using adenovirus vectors. Eliminating the Src-binding site on Pyk2 (Pyk2(Y402F)) markedly inhibited bone resorption by osteoclast-like cells, whereas kinase-dead Pyk2 had little effect. Kinase-dead Src, unlike kinase-dead Pyk2, markedly inhibited the bone-resorbing activity of wild type osteoclasts and failed to significantly restore bone-resorbing activity to Src(-/-) osteoclast-like cells. Activation of Src kinase by overexpressing kinase-dead Csk failed to reverse the inhibitory effect of Pyk2(Y402F), suggesting that osteoclastic bone resorption requires both c-Src kinase activity and the targeting of Src kinase by Pyk2. Src-catalyzed phosphorylation of Cbl on Tyr-731 is reported to induce the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. Expressing the Cbl(Y731F) mutant in osteoclasts markedly reduced their bone resorbing activity, suggesting that phosphorylation of Cbl(Y731) and the subsequent recruitment and activation of phosphatidylinositol 3-kinase may be critical signaling events downstream of Src in osteoclasts.     

Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species

c-Jun NH(2)-terminal kinase (JNK) is activated by a number of cellular stimuli such as inflammatory cytokines and environmental stresses. Reactive oxygen species also cause activation of JNK; however, the signaling cascade that leads to JNK activation remains to be elucidated. Because recent reports showed that expression of Cas, a putative Src substrate, stimulates JNK activation, we hypothesized that the Src kinase family and Cas would be involved in JNK activation by reactive oxygen species. An essential role for both Src and Cas was demonstrated. First, the specific Src family tyrosine kinase inhibitor, PP2, inhibited JNK activation by H(2)O(2) in a concentration-dependent manner but had no effect on extracellular signal-regulated kinases 1 and 2 and p38 activation. Second, JNK activation in response to H(2)O(2) was completely inhibited in cells derived from transgenic mice deficient in Src but not Fyn. Third, expression of a dominant negative mutant of Cas prevented H(2)O(2)-mediated JNK activation but had no effect on extracellular signal-regulated kinases 1 and 2 and p38 activation. Finally, the importance of Src was further supported by the inhibition of both H(2)O(2)-mediated Cas tyrosine phosphorylation and Cas.Crk complex formation in Src-/- but not Fyn-/- cells. These results demonstrate an essential role for Src and Cas in H(2)O(2)-mediated activation of JNK and suggest a new redox-sensitive pathway for JNK activation mediated by Src.     

Functional activation of Src family kinase yes protein is essential for the enhanced malignant properties of human melanoma cells expressing ganglioside GD3

The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3+). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3+ cells than in GD3- cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3+ cells than in GD3- cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipid-enriched microdomain/rafts in GD3+ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3- cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3- cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.     

SRC catalytic but not scaffolding function is needed for integrin-regulated tyrosine phosphorylation, cell migration, and cell spreading

Src family kinases (SFKs) are crucial for signaling through a variety of cell surface receptors, including integrins. There is evidence that integrin activation induces focal adhesion kinase (FAK) autophosphorylation at Y397 and that Src binds to and is activated by FAK to carry out subsequent phosphorylation events. However, it has also been suggested that Src functions as a scaffolding molecule through its SH2 and SH3 domains and that its kinase activity is not necessary. To examine the role of SFKs in integrin signaling, we have expressed various Src molecules in fibroblasts lacking other SFKs. In cells plated on fibronectin, FAK could indeed autophosphorylate at Y397 independently of Src but with lower efficiency than when Src was present. This step was promoted by kinase-inactive Src, but Src kinase activity was required for full rescue. Src kinase activity was also required for phosphorylation of additional sites on FAK and for other integrin-directed functions, including cell migration and spreading on fibronectin. In contrast, Src mutations in the SH2 or SH3 domain greatly reduced binding to FAK, Cas, and paxillin but had little effect on tyrosine phosphorylation or biological assays. Furthermore, our indirect evidence indicates that Src kinase activity does not need to be regulated to promote cell migration and FAK phosphorylation. Although Src clearly plays important roles in integrin signaling, it was not concentrated in focal adhesions. These results indicate that the primary role of Src in integrin signaling is as a kinase. Indirect models for Src function are proposed.     

Cbl associates with Pyk2 and Src to regulate Src kinase activity, alpha(v)beta(3) integrin-mediated signaling, cell adhesion, and osteoclast motility

The signaling events downstream of integrins that regulate cell attachment and motility are only partially understood. Using osteoclasts and transfected 293 cells, we find that a molecular complex comprising Src, Pyk2, and Cbl functions to regulate cell adhesion and motility. The activation of integrin alpha(v)beta(3) induces the [Ca(2+)](i)-dependent phosphorylation of Pyk2 Y402, its association with Src SH2, Src activation, and the Src SH3-dependent recruitment and phosphorylation of c-Cbl. Furthermore, the PTB domain of Cbl is shown to bind to phosphorylated Tyr-416 in the activation loop of Src, the autophosphorylation site of Src, inhibiting Src kinase activity and integrin-mediated adhesion. Finally, we show that deletion of c Src or c-Cbl leads to a decrease in osteoclast migration. Thus, binding of alpha(v)beta(3) integrin induces the formation of a Pyk2/Src/Cbl complex in which Cbl is a key regulator of Src kinase activity and of cell adhesion and migration. These findings may explain the osteopetrotic phenotype in the Src(-/-) mice.     

4-Anilino-3-cyanobenzo[g]quinolines as kinase inhibitors

A series of 4-anilino-3-cyanobenzo[g]quinolines was prepared as potent kinase inhibitors. Compared with their bicyclic 4-anilino-3-cyanoquinoline analogues, the tricyclic 4-anilino-3-cyanobenzo[g]quinolines are less active against EGF-R kinase, equally active against MAPK kinase (MEK), and more active against Src kinase. For Src kinase inhibition, the best activity is obtained when both the 7- and 8-positions are substituted with alkoxy groups. Several of these kinase inhibitors show potent growth inhibitory activity in tumor cells.